Antisense oligodeoxynucleotides to bax mRNA promote survival of rat sympathetic neurons in culture

Abstract

Previous in vitro studies have shown that the presence of high levels of Bax protein accelerated the rate of cell death following growth factor deprivation and that the ratio of cell death repressor Bcl-2 to cell death effector Bax may determine the susceptibility to apoptosis. Both Bcl-2 and Bax protein expression has been detected in sympathetic neurons in vivo, and overexpression of bcl-2 in cultured sympathetic neurons prevented apoptosis after deprivation of nerve growth factor (NGF). In the present study, we investigated the expression of bax and bcl-2 in primary cultures of sympathetic neurons from rat superior cervical ganglia. Furthermore, we tested the effects of a partially phosphorothioated bax antisense oligodeoxynucleotide (ODN) on the survival of sympathetic neurons in cultures supplied with suboptimal concentrations of NGF (0.5 ng/ml). A constitutive expression of bax mRNA at high levels was detected by reverse transcription and polymerase chain reaction which did not change significantly following NGF reduction or treatment with bax antisense ODN. A decrease in Bcl-2 immunoreactivity was observed by immunocytochemistry in tyrosine hydroxylase-positive neurons when cultured under suboptimal NGF concentrations, whereas Bcl-2 immunolabeled non-neuronal cells were not affected. Maximal number of neurons was obtained in control cultures containing 50 ng/ml of NGF. Few neurons survived in cultures grown in 0.5 ng/ml of NGF for 2 days (12.0 +/- 1.5% of controls, mean +/- SEM). Addition of two control ODNs at 1 microM had no effect on neuronal survival (10.1 +/- 1.2% and 11.0 +/- 1.3%, respectively), while the number of neurons was significantly increased in NGF-reduced cultures treated with a bax antisense ODNs (1 microM) (31.5 +/- 1.9%). Administration of fluorescein-labeled ODNs demonstrated intracellular uptake into cultured neurons. Treatment with bax antisense ODNs caused a significant reduction of Bax protein levels in SCG neurons by 46 +/- 2.6% as assessed by immuno-cytochemistry and digital image analysis. Taken together, our data demonstrate a constitutive expression of bax mRNA in sympathetic neurons suggesting that activation of bax expression may not be required for neuronal cell death after NGF withdrawal. After changing to suboptimal NGF concentrations, the cell-specific reduction in Bcl-2 immunoreactivity preceded morphological signs of degeneration indicating that growth factor starvation may down-regulate neuronal bcl-2 expression. Treatment with bax antisense ODNs indicated that suppression of Bax protein synthesis may promote neuronal survival in the threshold situation of insufficient trophic support.

Antisense Inhibition of Bax mRNA Increases Survival of Terminally Differentiated HL60 Cells

“…These results are in good agreement with the data of Bax-AS inactivation in rat symphathetic neurons in culture, where promotion of survival occurs (Gillardon et al, 1996). Our data clearly indicate that the bax gene plays an important role in activation of the genetic program underlying cell death in myeloid cells and that it is not efficiently complemented by other proapoptotic members of the Bcl-2 family, although they are expressed in this cellular context .…”

Up‐regulation of Bax Protein in Degenerating Retinal Ganglion Cells Precedes Apoptotic Cell Death after Optic Nerve Lesion in the Rat

“…It will be important to define, with a more refined methodology, if certain subtypes of RGCs display differential susceptibility to apoptosis and if this is correlated with Bax expression patterns. Interestingly, inhibition of Bax protein synthesis by antisense oligonucleotides increased cell survival at suboptimal nerve growth factor concentrations in primary sympathetic neurons (Gillardon et al, 1996), and sympathetic neurons derived from bax-deficient mice are resistant to growth factor deprivation (Deckwerth et al, 1996).…”

Bax is essential for mitochondrion-mediated apoptosis but not for cell death caused by photodynamic therapy

“…The 3 0 -oligonucleotides were biotinylated to facilitate the monitoring of intracellular uptake. The sequences employed were as follows: AS 1: 5 0 -TGCTC-CCGGACCCGTCCAT-3 0 (Gillardon et al, 1996); scrambled 1: 5 0 -TCATCGCTGGTAGAACACCT-3 0 ; AS 2: 5 0 -TCGATCCTGGAT-GAAACCCT-3 0 (Dibbert et al, 1999); scrambled 2: 5 0 -TGGTCCC-GCTCCCGCCACAT-3 0 .…”

“…Two Bax AS oligonucleotide sequences, previously reported to suppress Bax expression (Gillardon et al, 1996;Dibbert et al, 1999), were used. Since no significant difference was found between the two sequences with regard to the suppression of either Bax protein level or apoptosis, the results were pooled, and in later experiments only AS 1 was used.…”